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smear in gel electrophoresis after PCR

PCR

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#1 shawn6022

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Posted 24 July 2013 - 04:58 PM

Hello,
I have a problem with my PCR. I have been able to crossover PCR to get a deletion construct and ligate it into the vector to make the mutant. I then electroporated this vector into my wild type Ps. aeruginosa PAO1 and got colonies on the antibiotic selection plate. Then I patch plated these onto antibiotic plates and LB-no salt-10% sucrose to screen for merodiploids. The colonies that grew on the antibiotic and not on the sucrose (well diminished growth on sucrose) were then screened by colony PCR. The problem I am having is that everytime I run the PCR product on the gel I get a smear going from the top well to the bottom of the gel. I am using the same conditions that I used to construct the knockout mutant.

any suggestions/advice would be greatly appreciated!

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#2 phage434

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Posted 25 July 2013 - 06:23 AM

Your colony PCR is simply not working. There can be many reasons. Most common is too many cells. Use a 20 ul tip and put a very small amount of colony in a 50 ul water sample, vortex or mix well, and use on 1-2 ul in a pcr reaction. You could also verify that your primers and pcr reaction conditions worked by isolating gDNA from a sample of cells to make sure there was not some other issue. The colony pcr also typically includes a 5 minute initial 95-98 denature/cell lyse phase of the pcr reaction.

#3 shawn6022

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Posted 25 July 2013 - 08:12 AM

I think the issue might be that I am adding too many cells..I tried the reaction with isolated gDNA same conditions and got a nice band at the correct bp..I think I am going to try this method you said. I will post again after. Thanks!





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